The long term objective of this proposal is to understand the mechanisms involved in the differentiation of embryonic ectoderm in sea urchin development. The sea urchin Spec genes are the main focus of the proposed experiments. The Spec1 and Spec2 genes encode a family of calcium binding proteins whose accumulation is restricted to aboral ectoderm lineages. Spec3 encodes a protein hypothesized to be associated with ectodermal ciliogenesis.
The specific aims are: (1) To investigate the mechanisms by which Spec1 and Spec2 genes are transcriptionally activated in aboral ectoderm cells. DNA sequences involved in expression will be identified using the sea urchin embryo expression system. Regions required for appropriate expression will be assayed for specific protein interactions using gel mobility shift and DNA footprinting techniques. Comparisons will be made among the different Spec genes and between the Spec genes and the CyIIIa actin gene whose expression is also limited to the same cells; (2) To obtain information on the properties of the Spec1 protein. Purified Spec1 protein will be used to determine calcium binding affinities. Interactions of Spec1 with other proteins will be examined by gel transfer techniques. The hypothesis that the Spec1 protein interacts with microfilaments will be tested by injection of FITC-labeled Spec1 protein into sea urchin eggs and observing whether the protein is localized to the cortex or cleavage furrows. The Spec1 protein also will be produced in mouse C2 myoblasts and its intracellular sites monitored by indirect immunofluorescence; (3) To compare the Spec1 gene of S. purpuratus to the homologous gene of its distant relative, L. pictus. Conserved sequences important for proper function and expression will be sought. The Spec1 genes will be compared by microinjection of the genes into homologous and heterologous eggs and monitoring proper expression; (4) To test the hypothesis that the Spec3 protein is associated with embryonic cilia and that its levels in the cell are coupled to levels of unpolymerized tubulin. Pools of unpolymerized tubulin will be increased by treating embryos with colcemid and Spec3 mRNA synthesis and stability will be monitored. Spec3 mRNA will be expressed in mouse L cells to determine whether the stability of the urchin mRNA is the same as endogenous mRNAs encoding microtubular components. Antibodies against the Spec3 protein will be generated from the Spec3 ORF and the location of the protein in urchin embryos will be determined by immunological detection.
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